ES2531447T3 - Implantable device with antibacterial properties and multifunctional surface - Google Patents

Abstract

Implantable device for implantation in a human or animal body, in which at least a part of the surface of said device is coated with a complex of hyaluronic acid and glycopeptide antibiotic and in which said hyaluronic acid has an average molecular weight between 400,000 and 1,000,000 Da.

Description

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DESCRIPTION

Implantable device with antibacterial properties and multifunctional surface

The present invention relates to an implantation device whose surface combines antibacterial activity to prevent periprosthetic infections and improved osseointegration capacity.

Background of the invention

In orthopedics, periprosthetic infection (IPP) is a devastating consequence of implant insertion. Currently, the incidence of PPI ranges from 1 to 5%, with even higher values for high-risk patients and for those who have suffered trauma. In addition, approximately 73% of operations reviews are carried out after a peri-implant bacterial infection. The social and economic cost of these interventions is considerably high. The etiology of IPP is complex and depends on the ability of bacterial species to elude the host tissue response, usually by the formation of a biofilm. Actually, once inserted, the implant device is coated by whey proteins. This process is followed by interactions with cell species and tissue regeneration or repair in cases with positive course. In the presence of bacterial species, the implant surface may undergo bacterial adhesion and biofilm formation. In particular, within the biofilm the bacteria are protected by the immunovigilance system and by the effect of systemic antibiotics. In this way, colonization can spread, with the known harmful consequences in the field of periprosthetic infection. A series of systems were developed for the local release of antibiotics, both from cements and micrometric layers of biodegradable polymers deposited on the surface of the devices, with the aim of fighting IPP. However, after the release is complete, porous systems of this type can serve as a protected site for adhesion

25 bacterial and biofilm formation.

Recently, literature articles have been submitted in which an antibiotic, vancomycin, covalently bound to the surface of implant devices made of titanium and conducted bactericidal activity against bacterial species belonging to the genus Staphylococci (Chemistry & Biology, vol. 12, 1041-1048, 2005, Vancomycin Covalently Bonded to Titanium Beads Kills Staphylococcus aureus; Journal Orthopedic Research, 25, 858-866, 2007, Vancomycin Covalently Bonded to Titanium Alloy Prevents Bacterial Colonization).

The immobilization of vancomycin on the surface of implant or local release devices thereof are, for these applications, processes of great interest. Vancomycin is a potent drug to treat infections by Gram-positive bacteria, which are considerably the most common causes of periprosthetic infections. The mechanism of action of vancomycin provides the blockade of the synthesis of the peptidoglycan layer of the cell walls of Gram-positive bacteria by means of the terminal L-Lys-D-Ala-D-Ala terminal of the nascent peptidoglycan. In this way, vancomycin prevents the crosslinking that is required for osmotic stability. The concept of vancomycin that binds firmly, in itself soluble in water, to the surface of implant devices exceeds the vision of the simple release system. Actually, in this case there is a high local concentration of a drug firmly attached to the interface between the implant tax and the external environment. This stable pharmacological barrier prevents the formation of bacterial colonies on the surface of the implant tax, thus preventing the appearance of IPP. As described by Chapiro and collaborators in the article Selfprotective Smart Orthopedic Implants, Expert Rev. Med. Devices, 2007 Jan; 4 (1): 55-64, systems of this

45 type can lead to a new generation of implant devices, which are self-protected against risks of bacterial infection due to their surface properties.

However, the process of joining vancomycin to the surface of the device made of titanium described in these articles comprises different stages, which are quite complex from a practical point of view and not easily adaptable to industrial production, which makes it unsuitable for devices of a given dimension and complex geometry.

MATSUNO HIROAKI Y COL .: "Antibiotic-containing hyaluronic acid gel as an antibacterial carrier: Usefulness of sponge and film-formed HA gel in deep infection", JOURNAL OF ORTHOPAEDIC RESEARCH, vol. 24, nº 3, 6 of

January 55, 2006 (06-01-2006), pages 321-326, discloses: an implantable titanium hip prosthesis to be implanted in the human body, in which the surface of said device is coated with an acid complex hyaluronic (HA) and a vancomycin (undefined). The HA is crosslinked and has a MW of 2000 kDa.

CHUANG H F Y COL .: “Polyelectrolyte multilayers for tunable release of antibiotics”, BIOMACROMOLECULES JUNE 2008 AMERICAN CHEMICAL SOCIETY US, vol. 9, nº 6, May 14, 2008 (05-14-2008), pages 16601668, reveals:

an implantable prosthesis to be implanted in the human being, in which the surface of said device is etched by plasma and then covered layer by layer with polyethylenediamine and a hyaluronic acid (HA) complex

65 of 1760 kDa and a gentamicin. The coating layer has a thickness of 680, 590 or 320 nm. Gentamicin is released in a controlled manner.

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Obviously, the provision, under conditions compatible with a productive context, of prosthetic implant devices that can exploit the pharmacological action of vancomycin would constitute a considerable step forward in the sector, with considerable scientific, social and economic implications. Ideally, the process could accompany the immobilization / release of vancomycin to provide other surface properties

5 considerable, such as increased osseointegration rate. In fact, the rapid regeneration of bone tissue, guaranteeing the occupation of the available surface of the implanted device, reduces the chances of superficial colonization by bacterial cells, completing the antibacterial protective effect due to vancomycin. This concept would allow the current provision of devices with multifunctional surfaces, that is, surfaces that also perform, in addition to the obvious function of supporting tissue components, for example:

-the function of stimulating tissue regeneration / repair

-The antibacterial protection function.

15 Studies carried out by the present inventor have now revealed the possibility of practically implementing the previously mentioned concepts as described herein below.

Summary of the invention

A first scope of the present invention is to provide an implantable device to be implanted in a human or animal body, in which at least a part of the surface of said device is coated with a complex of hyaluronic acid and glycopeptide antibiotic and in which said hyaluronic acid has an average molecular weight between 400,000 and 1,000,000 Da.

A second scope of the present invention is to provide a collagen gel comprising the hyaluronic acid complex with a glycopeptide antibiotic, as such or lyophilized, in which said hyaluronic acid has an average molecular weight between 400,000 and 1,000,000. Gives.

A third scope of the present invention is to provide a method for obtaining an implantable device according to the first embodiment or a gel according to the second embodiment, comprising the step of forming said hyaluronic acid complex with said glycopeptide antibiotic causing said hyaluronic acid to be contact a solution of said glycopeptide antibiotic, in the presence of a condensation or cross-linking agent.

The present invention considers the process of obtaining an implant tax in the human or animal body that can combine the immobilization of an antibiotic, in particular vancomycin, in a manner and effective, with the stimulation of osteogenic cells and guaranteeing the increase of osseointegration rate The rapid formation of bone tissue, with relative occupation of the available surface of the implanted device, reduces the chances of superficial colonization by bacterial cells and thus constitutes a synergistic effect with the antibiotic effect of vancomycin. According to the process object of the invention, devices with multifunctional surfaces are thus provided, that is, surfaces that exercise, in addition to the obvious function of supporting the tissue components, also the aforementioned functions of stimulating tissue regeneration / repair and antibacterial protection.

The present invention is based on the surprising observation that vancomycin, a water soluble compound, if present in an aqueous solution with the hyaluronic acid molecule in the presence of cross-linking / condensing agents, forms a compound / precipitate with said hyaluronic acid. From this observation, a process has been developed that reproduces these events on the surface of implant devices. The process provides, at the beginning, the linkage of the hyaluronic acid molecule with a properly functionalized surface (with methods known in the literature); then, the surface coated with hyaluronic acid is incubated in an aqueous solution of vancomycin in the presence of crosslinking agents in suitable concentration. Surprisingly, the formation of hyaluronic acid / vancomycin precipitates is observed gradually on the surface, also macroscopically observable due to the formation

A gradual “opaque” layer that coats homogeneously and covers the entire surface, an event that does not occur if the surface had not previously been coated with hyaluronic acid.

Without being limited to a particular theory, this behavior is considered to be at least partial due to the ionic interaction between the negative charges present in the hyaluronic acid molecule and the positively charged amino groups of vancomycin. However, since the formation of said opaque layer only occurs in the presence of given crosslinking agents, the interaction could also be due to different physical and chemical factors, unexpectedly related to the molecular structure of hyaluronic acid.

For brevity, in the present patent application, such precipitates or compounds of a glycopeptide antibiotic,

65 in particular vancomycin, and hyaluronic acid should be defined as "hyaluronic acid / glycopeptide antibiotic complexes" or specifically "hyaluronic acid / vancomycin complexes", without implying that the typical bond of

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a complex is necessarily formed.

In addition, it was surprisingly discovered that the use of different molecular weights of hyaluronic acid (HA) greatly influences the formation of said HA / vancomycin precipitate: in this case, it was observed that

5 coating a surface with HA with low molecular weights (between 5000 Da and 80000 Da) is not effective in "capturing", in the post-treatment stage, an amount of vancomycin molecules equivalent to that captured when the surface is coated with hyaluronic acid with higher molecular weight. In addition, the maximum yield in the capture process does not increase as the weight of hyaluronic acid increases, but reveals a "peak" or bell development.

In addition, the use of other polysaccharides, including heparin, chondroitin sulfate, chitosan, alginic acid, pectins, has the effect of reducing the amount of functional vancomycin that is bound on the surface (in some cases reducing its stability, in others the pharmacological activity).

Surprisingly, the compound / precipitate incorporated into the surface layer is released if exposed to physiological aqueous environments, preserving the pharmacological action of vancomycin. Total vancomycin release is not immediate, as would be expected due to the high solubility of vancomycin in aqueous solutions, but the effect continues for several weeks, offering a prolonged interfacial antibiotic coating.

The hyaluronic acid is deposited on the surface of the device to be coated using different methods that allow pretreating the surface to be coated with allylamine plasma or aqueous polyethyleneimine solution, with amino groups that can allow covalent bonding with the carboxyl groups of the hyaluronic acid.

According to a preferred embodiment, the surface to be treated can be coated with a single layer of collagen molecules, preferably in fibrillated forms: the amino groups of the amino acid residues of the collagen molecule can be attached to the carboxyl groups of the hyaluronic acid . The functionalization of the surfaces with collagen also allows to increase the "osteoinductive" properties of the surface: in fact, it was observed that such modification can direct the differentiation of human mesenchymal cells towards the osteogenic line.

The process described above is versatile and is not limited by the type of device. It can thus be applied both on metal devices such as screws made of titanium to fix fractures, for prostheses, on polymeric devices, both biodegradable and permanent, and on ceramic devices, for example, hydroxyapatite or other forms of calcium phosphates, even in the form of powder and particle-like granules such as

35 is used for bone cavity fillings.

The process of the invention can be applied to molded bodies based on natural materials such as sponges or scaffolds based on collagen or natural polymers or ceramic materials. It can also be applied to suspensions of nanostructured organic material, such as collagen fibrils with a smaller diameter of a micrometer.

In the case of application to collagen suspensions or nanostructured suspension materials, the interaction object of the present invention leads to the formation of a gel. This gel - based on collagen and containing vancomycin and hyaluronic acid - can be used as such or in lyophilized forms and reconstituted if used as

Filling material at the fixation site of an implantable device, performing both the typical osteointegration action of collagen and hyaluronic acid and the antibacterial activity of the antibiotic also in the region surrounding the implant.

Thus, these processes allow to provide a coating of vancomycin in hyaluronic acid on the surface of the devices or materials, implementing the concept of slow release pharmacological barrier to the device / tissue interface. In addition, surface modification of implant devices with hyaluronic acid increases the tendency of the osseointegration material, as described, for example, by Morra et al. in the article Covalently-Linked Hyaluronan Promotes Bone Formation around Ti Implants in a Rabbit Model, published in the Journal of Orthopedic Research, 27: 657-663, 2009 and in patent application WO 2006/038056 A1.

Thus, the previously described methods allow to provide implant devices that not only have optimal and improved osseointegration characteristics, but also provide the self-protective and bactericidal characteristics of vancomycin, practically translating the multifunctional surface concept. In addition, the method of the present invention allows prolonging the multifunctionality of the surface, exemplified by the simultaneous presence of the bioactive chemical composition with osteogenic action (hyaluronic acid bond) and pharmacological action (vancomycin release), adding the possibility of controlling the surface topography through roughing processes. With respect to this, it should be taken into account that in the field of dental implants the osseointegration process is facilitated by the action of surface topography on osteogenic cells, as exemplified by the concept of “Modulation of osteogenesis via implant surface

65 design ”, described by Boyan, B.D., Schwartz, Z., in: Davies J. E. editor. Bone Engineering, Toronto, EM Squared, 232-239, 2000. This concept has been so satisfying to the point that dental implants with a smooth surface

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They are no longer available in the market, all having a rough surface topography in place. In orthopedics, for example in the fixation of fractures by means of screws made of titanium, this concept could not be exploited due to the fear of offering, by roughing the surface, surfaces that could be exploited for adhesion and bacterial colonization. The present invention allows instead to provide a surface osteogenic layer of

5 conforming hyaluronic acid (that is, it does not alter the surface topography, adapting to it), which can also exploit the roughness of the surface topography in which vancomycin release prevents bacterial colonization of irregularity and roughness of the surface.

Description of the invention

The present invention considers in particular an implantation device in the human or animal body, in which at least a part of the surface of said device is coated with a "hyaluronic acid compound with a glycopeptide antibiotic.

In one embodiment, the glycopeptide antibiotic is vancomycin.

The idea is to bind a layer of hyaluronic acid to the surfaces of interest (by methods known in the art), then incubate such surfaces with a vancomycin solution in the presence of condensing agents, in particular N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), present simultaneously: an "opaque" layer is formed, indicating the interaction between previously bound hyaluronic acid and vancomycin on the surface after a few hours. This opaque layer is not formed in the absence of surface-bound, functionalized or non-functionalized hyaluronic acid, and is not formed in the absence of NHS / EDC. Hereafter, reference should be made to "hyaluronic acid complexes with vancomycin", in which the term "complex" comprises any type of chemical / physical or simply physical interaction between the acid

Hyaluronic and vancomycin, which includes hydrogen bond interactions, and / or van der Waals interactions and / or electrostatic interactions, etc., which are obtained by vancomycin precipitation or co-precipitation of hyaluronic acid and vancomycin in the presence of crosslinking or condensing agents and in particular conditions (pH and concentration of the solutions).

In one embodiment, the entire surface of the implantation device is coated with said glycopeptide hyaluronic acid / antibiotic complex bound to the surface having a. Alternatively, part of the device is coated with hyaluronic acid, and the formation of vancomycin complexes only occurs in this area.

The implantation device according to the invention can be made of metal (for example, steel, titanium or

35 alloys thereof with other metals) or made of plastic material (such as, for example, polystyrene) compatible for applications on the human or animal body, or made of ceramic material, both in the form of the device as granulate / powder.

In one embodiment, the device consists of a dental implant screw, preferably made of titanium or alloys thereof, possibly of the transmucosal type, or a screw, preferably made of titanium or alloys thereof, for spinal or skeletal fixation, or for the fracture fixation, or an intervertebral disc, preferably made of titanium, alloys thereof or cobalt-chromium alloys or made of metal alloys commonly used for these applications. The surface of the device made of titanium can be smooth (commonly referred to as "carved") or preferably roughened according to methods known in the art, in

Particularly by sandblasting, using alumina, titanium oxides or other sandblasting agents, roughing treatment using acids, or electrochemical roughing treatment.

In one embodiment, the process of immobilization of the hyaluronic acid complex with the glycopeptide antibiotic on an implantable device according to the invention provides the introduction of functional amino groups on the surface of the device. Hyaluronic acid covalently binds to surface amino groups, by methods known in the art, and exerts its action of "capturing" vancomycin with complex formation in the presence of EDC and NHS in the next stage. The average molecular weight of hyaluronic acid comprises between 400,000 and 1 million Da. The hyaluronic acid bonding with the aminated surface can be produced by methods known in the art, for example, using EDC / NHS, or treating acid

Hyaluronic acid with periodic acid and subsequent reductive amination, or as described in WO 2006/038056 A1. The layer thus obtained can have a thickness between 0.1 and 1000 nanometers, preferably between 1 and 20 nm.

The amino groups can be deposited on the surface of the implantable device according to methods widely known in the sector. The technique that provides the introduction of the substrate having functional amino groups on the surface of the implantable device by plasma deposition of molecules carrying the amino groups is particularly advantageous. Typical examples of molecules used for this purpose are allylamine, alkylamines such as hexylamine or heptylamine and, generally, organic molecules having amino functions that exhibit the volatility characteristics required in the plasma state. Plasma deposition of 65 amine occurs under the following conditions: pressure between 80 and 300 mTorr, discharge power between 5 and 200 W, deposition time between 1 ms and 300 s. Plasma deposition can also

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occur under conditions of pulsed plasma, with active and inactive plasma cycles between 1 and 100 ms, to minimize molecular fragmentation and maintain the highest possible density of the amino groups. Plasma deposition treatment of amines may be preceded by other plasma treatments, for example, using air plasma or oxygen to clean the surface and increase adhesion with the substrate.

5 Another method of coating the implantable device with a substrate containing amino groups consists of adsorbing polyethyleneimine (PEI) on the surface of the device, for example, of a 0.2% aqueous solution, for 2 hours, at room temperature. Also the use of silanes, for example 3-aminopropyltriethoxysilane (APTES), or analogous compounds, is within the common methods of surface functionalization.

It is also possible to functionalize the surface of the implantable device with carboxyl groups by means of plasma deposition of acrylic / methacrylic acid.

In addition, it is possible to functionalize the surface with natural, possibly bioactive, molecules that are

They are irreversibly adsorbed on the surface and have suitable amino / carboxylic groups for subsequent hyaluronic acid bonding and complexes thereof with vancomycin. Typical examples found within this category are collagen and other extracellular matrix protein molecules, such as laminin, fibronectin, vitronectin. According to this embodiment, the implantation device according to the invention comprises a first layer of adsorbed collagen, in a monomer or fibrillated form, to which hyaluronic acid is attached, according to the process described, preferably by EDC / NHS, or by reductive amination. This is followed by the formation of hyaluronic acid / vancomycin complexes, by exposure to vancomycin and solutions of crosslinking or condensing agents. Collagen functionalization is preferably performed with a 0.3% solution of collagen in 10 mM acetic acid and an equal volume of phosphate buffer at 37 ° C for 8 hours.

The previously described method can also be carried out on suspended collagen, leading to the formation - in the aqueous medium - of a precipitate of fibrillated collagen complex, hyaluronic acid and glycopeptide antibiotic (such as vancomycin), which can be separated, washed and partially dry to obtain a gel. This gel based on collagen and containing said antibiotic of glycopeptide and hyaluronic acid can be used as such or in lyophilized forms and can be reconstructed if used, as filler material at the fixation site of said implantable device, performing both the typical osseointegration action of collagen and hyaluronic acid, in addition to the antibacterial activity of the antibiotic also in the region surrounding the implant.

Another object of the invention is a kit comprising an implantation device on the human body or

An animal, wherein at least a portion of the surface of said device is coated with a hyaluronic acid complex with a glycopeptide antibiotic, and a fibrilated collagen gel with said glycopeptide hyaluronic acid / antibiotic complex.

The following examples describe the invention.

Example 1. Interaction of hyaluronic acid-vancomycin.

This example shows that there is a surprising interaction between vancomycin and hyaluronic acid.

A 0.1% solution (weight / volume) of HW hyaluronic acid (molecular weight 800 kDa, Lifecore) in milliQ water and a 0.5% solution of vancomycin in milliQ water is prepared. The hyaluronic acid solution is gradually added to the last solution and a gradual increase in the turbidity of the solution is observed until the formation of suspended particles (sign of strong interaction between HA and vancomycin). If two condensing agents, N-hydroxysuccinimide and subsequently ethyl carbodiimide, are added to the mixture, the solution becomes instantly transparent. However, after 15 hours, the mixture becomes very cloudy, with the presence of suspended precipitates: condensing agents can probably bind vancomycin amino groups to hyaluronic acid carboxyl groups. Such precipitates were collected, washed with milliQ water and subsequently analyzed under IR: Figure 1 shows the spectra with respect to vancomycin (dots), hyaluronic acid (dashes) and the precipitate formed by the described method (continuous). In this

There are signs of vancomycin and hyaluronic acid signals, which confirm the interaction between the two molecules.

Example 2. Interaction of hyaluronic acid-vancomycin.

This example shows the fundamental role of condensing agents in the interaction between vancomycin and hyaluronic acid.

The previous experiment is repeated using solutions of hyaluronic acid and vancomycin at very low concentrations (up to 10 times lower): in these cases, the addition of hyaluronic acid to a solution of vancomycin no

65 produces an immediate turbidity of the mixture, which remains transparent. However, adding the same amounts of condensing agents used in the previous experiment and leaving in incubation for

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fifteen

25

35

Four. Five

55

approximately 15 hours, the solution becomes cloudy (although to a lesser extent with respect to the large precipitates formed in the previous experiment), demonstrating that the interaction of HA-vancomycin is somewhat "stimulated" by the presence of condensing agents.

Solutions with high concentration of vancomycin in water (1%) and HA in water (0.5%) were prepared in order to observe possible differences between the interaction of HA-vancomycin that occurs in the presence or absence of condensing agents . Such solutions were mixed and the immediately formed precipitate was collected, washed and analyzed under IR. The spectrum was then compared with respect to this precipitate with that with respect to the precipitate that is formed in the presence of condensing agents.

Figure 2 shows - through a dotted and continuous line - the spectra of HA and vancomycin respectively, while the long dashed line shows the spectrum of the HA-vancomycin precipitate in the presence of condensing agents and the short dashed line shows the spectrum of the precipitate in the absence of condensing agents.

There are two important signals present in the spectrum in the long dashed line and not in the short dashed line: the first is at 1550 cm-1, which could be the signal of the secondary amide (torsion NH2 and CN voltage); the second is at 840 cm-1 and could be the signal of aliphatic amines and OCN torsion. These two signals, only present in the spectrum of the precipitate formed in the presence of condensing agents (but which do not really belong to the condensing agents), could indicate different links from those derived from simple electrostatic interaction, such as bonds covalent, specific interactions or other interactions formed in such precipitate.

The possible existence of a strong interaction between hyaluronic acid and vancomycin has paved the way for new surface modification experiments. Such experiments consist of covalently binding the hyaluronic acid to a suitably functionalized surface, then incubating the aforementioned surface in a vancomycin solution in the presence of condensing agents, so as to directly provide the same reaction observed in the solution on the surface.

Example 3. Interaction of HA-vancomycin on a surface.

This example shows that the process observed in solution also occurs directly on the surface of a material.

Polystyrene surfaces were prepared as follows:

-treatment with air plasma for 20 seconds

-incubation with 0.5% polyethyleneimine (PEI) solution in water for 2 hours

- washed with milliQ water (3 times)

- Incubation of the polystyrene surfaces overnight with 800% Da Life Solution of 800 kDa hyaluronic acid in milliQ water, in the presence of 5 mg / cm3 of NHS and 7.5 mg / cm3 of EDC

- washed with milliQ water (2 times)

- Incubation with 0.7% solution of vancomycin in water overnight, in the presence of 5 mg / cm3 of NHS and 7.5 mg / cm3 of EDC.

- washed with milliQ water (3 times)

At the end of the treatment on the polystyrene surfaces thus prepared, a very homogeneous opaque thin layer is observed, derived from the interaction produced between the hyaluronic acid bound on the surface in the first reaction stage and the vancomycin added in the second reaction stage. The polystyrene surfaces treated in the same way, but in the absence of NHS and EDC during the second incubation in vancomycin solution, are perfectly transparent, hence the importance of the condensation agents is confirmed.

Example 4 - Coating process of an implant screw made of titanium with hyaluronic acid and vancomycin.

Some implant screws made of titanium, with a length of 13 mm and width of 4 mm, are treated as follows:

Sample 1: no treatment (Ti sample)

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Sample 2: surface functionalization using PEI, and HA binding as in Example 3 (Ti-HA sample).

Sample 3: A sample treated as in point 2 was subsequently incubated with a 0.7% solution of vancomycin in water overnight, in the presence of 5 mg / cm3 of NHS and 7.5 mg / cm3 of EDC, and subsequent washing with milliQ water (sample of Ti-HA-VXL)

The samples thus obtained were subsequently subjected to XPS analysis (X-ray photoelectronic spectroscopy) to evaluate the chemical composition of the surface. A sample of vancomycin was obtained, obtained

10 allowing a solution of vancomycin to evaporate in milliQ water on a plastic substrate along it as a reference. The following results were obtained, expressed as a percentage of atoms and remembering that the XPS analysis does not measure the presence of hydrogen atoms:

Sample

C OR N You Cl

Vancomycin

77.8 15.9 4.6 1.7

You

34.8 46.4 0.4 18.4

Ti-HA

69.0 24.4 6.3 0.3

Ti-HA-VXL

69.5 23.2 5.8 0.6 0.9

The vancomycin molecule is characterized, for analytical purposes with respect to this evaluation, by the presence of the Cl element (two atoms in a compound that also comprises O, C and N with molecular weight of approximately 1450 Da). The presence of Cl, at an atomic percentage of less than 2%, is actually shown by the XPS analysis, as indicated in the table, on the line indicated by the vancomycin analysis. The surfaces of the Ti screw and the Ti-HA screw are characterized by composition values in line with expectations,

20 as is observable from the bibliography of the sector.

The surface composition of the Ti-HA-VXL sample is different from that of Ti-HA due to the presence of Cl, hence the introduction of the vancomycin molecule on the surface.

Example 5 - Example of the importance of the molecular weight of hyaluronic acid on the release of vancomycin from modified surfaces by the present process.

Modified polystyrene surfaces with different molecular weights of HA were prepared and then with vancomycin solution in the presence of NHS and EDC to observe the specificity of the HA reaction

30 vancomycin HA LW (10 kDa), HA MW (approximately 70 kDa), HA HW (880 kDa) and HA HHW (approximately 2000 kDa) were used. Thus, the surfaces were first functionalized with polyethyleneimine (0.5% solution in water for 2 hours); The incubation was then carried out overnight with the solutions of HA with different molecular weight in the presence of NHS and EDC. Subsequently the water wash was carried out and then the surfaces were incubated overnight with a 0.8% solution of vancomycin in water in

The presence of 5 mg / ml of NHS and 7.5 mg / ml of EDC.

The surface opacity was observed only with the HW weight of HA, although for HA MW and HA HHW the wells did not seem perfectly transparent: thus, there is a certain specificity, although only dimensional, in the interaction between vancomycin and HA HW.

At the end of the wash, the surfaces were incubated in PBS to perform vancomycin release analysis over time by HPLC. The diagram in Figure 3 shows the amount of vancomycin released by the different surfaces over time (one month): it can be seen that the surface modified with HA HW releases a greater amount of antibiotic with respect to those modified with the other molecular weights.

45 Example 6 - Influence of the functionalization process on the amount of vancomycin released by surfaces modified by the present process.

The treatment of two 6-well polystyrene plates with HA + VXL (VXL = vancomycin and agents) was planned.

50 EDC-NHS condensation), but functionalizing the surfaces with PEI (polyethyleneimine) or with fibrillated collagen to verify if the functionalization with fibrillated collagen produces some change in the vancomycin release rate of the polystyrene surfaces. The 6-well plates were treated with plasma as in the previous example, then the wells were incubated with a solution of 0.5% PEI in water for 2 hours or with a 0.3% solution of collagen in acetic acid 10 mM and an equal volume of PBS at 37 ° C for 8 hours.

Before the functionalization stage, the surfaces were treated with a 0.2% solution of HA HW in the presence of NHS and EDC overnight, then washed with milliQ water and incubated with a 0.75 solution % vancomycin in water in the presence of NHS and EDC overnight. At the end of such incubation, the surfaces were washed with milliQ water and dried.

60

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The release stage was performed in PBS at 37 ° C, with the measurement of the amount released at 4 hours, 24 hours, 4 days, 12 days and 21 days by HPLC. All times revealed that collagen modified surfaces release a greater amount of antibiotic compared to those functionalized with PEI. The diagram in Figure 4 shows the cumulative curve of vancomycin release from differently functionalized surfaces:

5 it can be observed that, at each moment of time, the surfaces functionalized with fibrillated collagen release a greater amount of vancomycin.

This example indicates that, by surprise and not completely clear reasons, the surface functionalization stage, in particular the molecular species used for surface functionalization, influences the total amount of

10 "captured" vancomycin from the surface coated with hyaluronic acid and / or the amount of vancomycin that the surface coated with hyaluronic acid can release. Going beyond the importance of this observation from a functional point of view, the result confirms that the observed phenomenon is not a general effect of the action of EDC-NHS on vancomycin in solution, but it is united in a surprising and unexpected way to the molecular structure of the surface.

15 Example 7 - Verification of the improved properties of stimulation of osteogenic behavior of mesenchymal cells.

In order to evaluate the response of mesenchymal cells to the coating process and thus verify if

The latter may also have effects on osteogenesis, mesenchymal cells were cultured on the following surfaces, provided on plates with microwells for cell cultures:

-plastic for cell cultures (control)

25 -plastic for cultures functionalized with PEI and subsequent covalent bond of 800 kDa hyaluronic acid (HA)

-plastic for PEI functionalized cultures and subsequent 800 kDa hyaluronic acid covalent bond, followed by HA-vancomycin complex formation as described in the previous example (HA-VXL)

30 Human bone marrow mesenchymal cells from Lonza Milano srl were acquired in an undifferentiated form. As is known, according to external stimuli, these cells can differentiate along some different trajectories, which include the osteogenic. The cells were cultured in an osteogenic medium and the expression of some genes responsible for the formation of bone tissue thereof was evaluated by real time polymerase chain reaction analysis (RT-PCR). The data is reported in the following table, in which "=" means

35 expression equivalent to that of the control, "+" expression up to 5 times greater than that of the control, "++" expression greater than 5 times that of the control.

The following results were obtained, after 10 days of culture:

Gen

HE HAS HA-VXL

Alkaline phosphatase

= +

Runx2

+ +

Osteocalcin

+ +

Bone sialoprotein

++ ++

Bone morphogenetic protein-2 (BMP-2)

++ ++

40 These data confirm that the coating with HA stimulates the expression of genes linked to bone tissue formation, confirming the in vivo data cited in the literature regarding the effect of covalent bonding of surface layers of HA on osseointegration (Morra et al. , Covalently-Linked Hyaluronan Promotes Bone Formation around Ti Implants in a Rabbit Model, published in the Journal of Orthopedic Research, 27: 657-663, 2009). In

In particular, both the RunX2 transcription factor, which controls cell differentiation, and in particular the BMP-2 and BSP protein (bone sialoprotein) express significantly in excess over HA and HA-VXL with respect to the control, and indicate a process Extremely significant osteogenic. The presence of vancomycin complexes does not substantially alter the advantages of HA (on the contrary, another very important gene, alkaline phosphatase, seems to express more about HA-VXL with respect to HA), confirming that this type of

50 surface is pro-osteogenic. This is a very important property, which combined with the peculiar release of the antibiotic, is at the base of the generation and design of multifunctional devices.

Example 8 - Verification of the antibacterial efficiency of implant screws obtained according to the present process.

Aura inhibition tests were performed on cultures of Staphylococcus epidermidis in order to demonstrate the antibacterial action of the present invention. Implants coated with HA and HA-VXL are provided by the methods described previously. Next, these implants, together with the negative controls (untreated implants), were incubated in a semi-solid agar medium together with the bacterial mixture in

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a Petri dish at 37 ° C. Bacteria should proliferate and, in the case of a surface with antibacterial properties (with antibiotic release), an inhibition halo should be formed, that is, an area without bacteria that surrounds the implant, around the implant. The results of this experiment confirmed that the treatment developed has antibacterial activities: in reality, around HA + VXL implants are

5 observes the inhibition halo, which does not form around the control implants. Figure 5 represents an image that explains the experiment, with the control implants in the upper part (only titanium on the left, Ti coated with HA on the right) and those treated in the lower part (HA-VXL, two replicas) , around which the inhibition halo is clearly observed.

10 The implants thus treated were subsequently collected and once again immersed in an agar containing bacteria to verify if they would maintain their antibacterial activity: in fact, the inhibition halo is formed around the implants treated in this second incubation, even having a slightly smaller diameter than the first time. The same also happened in the case of a third incubation.

Thus, the surfaces of the HA-VXL implants reveal the greatest osteogenic characteristics of hyaluronic acid, as shown by Example 7, with which the antibacterial properties revealed by the results exemplified by the photograph indicated in Figure 5 are combined. Thus, this example confirms the multifunctional nature of the surface obtained according to the present process, and the functional advantage thereof with respect to the conventional device (screw made of titanium) and with respect to the HA coated device described in the

20 subject

Example 9 - Making a fracture fixation screw made of titanium, with a rough surface and coating with hyaluronic acid-vancomycin.

25 A screw made of grade 5 titanium is used for fracture fixation to demonstrate the preparation of an implant device with multifunctional surface, which has the following properties:

-Rough surface and that guarantees the increase of the surface area

30 - bioactive surface by means of bonding with hyaluronic acid after functionalization by adsorption of fibrillated collagen

-Antibacterial surface by releasing vancomycin present in complexes with hyaluronic acid attached to the surface.

The apical portion (head) of the screw is produced by masking and the screw is subjected to a sandblasting process for 40 seconds in a Norblast sandblasting machine, using titanium oxides as a sandblasting agent. Next, the screw undergoes an acid treatment process, according to protocols commonly used by this company to treat dental implants, and then undergoes

The process in question as in Example 7 and 8.

The rough surface of the screw has a surface area, and thus a contact surface, more than 70% higher than that of a conventional screw.

Claims (17)

1.-Implantable device for implantation in a human or animal body, in which at least a part of the surface of said device is coated with a complex of hyaluronic acid and glycopeptide antibiotic and 5 in which said hyaluronic acid has a weight Average molecular weight between 400,000 and 1,000,000 Da. 2. Implantable device according to claim 1, wherein said device is made of steel, titanium or alloys thereof with other metals or plastic material, which are suitable for applications on the human or animal body, or is made of materials ceramics, such as hydroxyapatite or other forms of calcium phosphates, even in the form of particles-like powders and granules used as fillers of bone cavities. 3. Implantable device according to claim 1 or 2, wherein said device consists of a dental implant screw, preferably made of titanium or alloys thereof, possibly of transmucosal type, or of a screw, preferably made of titanium or alloys. thereof, for spinal or skeletal fixation, or consists 15 in an intervertebral disc, preferably made of titanium, alloys thereof or cobalt-chromium alloys or made of metal alloys that are commonly used for these applications. 4. Implantable device according to any one of claims 1 to 3, wherein said glycopeptide antibiotic is vancomycin. 5. Implantable device according to claims 1 to 4, wherein said hyaluronic acid is immobilized on the surface of said device. 6. Device according to claim 5, wherein the surface of said device is aminated. 7. Device according to claim 5, wherein the surface of said device comprises carboxyl groups obtained by plasma deposition of acrylic / methacrylic acid. 8. Device according to claim 5, wherein the surface of said device is functionalized with collagen or extracellular matrix protein molecules, preferably chosen from laminin, fibronectin and vitronectin, in which collagen is adsorbed on the surface of said device in a monomeric or fibrillated form. 9. Device according to any one of claims 1 to 8, wherein said hyaluronic acid complex with A glycopeptide antibiotic can be obtained by treating said hyaluronic acid with a solution of said glycopeptide antibiotic in the presence of a condensing or cross-linking agent. 10. Device according to claim 9, wherein said condensing or crosslinking agent is N-hydroxysuccinimide / 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. 11.-Collagen gel comprising said hyaluronic acid complex with a glycopeptide antibiotic, as such or lyophilized, wherein said hyaluronic acid complex with said glycopeptide antibiotic is as defined in any one of claims 1 to 4 . 12. Gel according to claim 11, to be used as filler material around a bone prosthesis implant. 13.-Kit comprising an implantable device as defined in any one of claims 1 to 10 and a gel as defined in claim 11 or 12. 14. Method for obtaining an implantable device according to any one of claims 1 to 10 or a gel according to claim 11 or 12, comprising the step of forming said hyaluronic acid complex with said glycopeptide antibiotic causing said hyaluronic acid to be contact a solution of said glycopeptide antibiotic, in the presence of a condensation or cross-linking agent. 15. A method according to claim 14, wherein said condensing or crosslinking agent is N-hydroxysuccinimide / 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. 16. A method according to claim 14 or 15, wherein the surface of said implantable device is aminated beforehand by surface plasma deposition of an amine chosen from allylamine, hexylamine or heptylamine or by deposition of polyethyleneimine or acrylic / methacrylic acid solution or by treatment using silanes, such as 3-aminopropyltriethoxysilane. 17. Method according to claim 16, wherein said hyaluronic acid is immobilized on said surface 65 aminated by condensation with N-hydroxysuccinimide / 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, or by treatment of hyaluronic acid with periodate and subsequent reductive amination. eleven 18. Method according to claim 14 or 15, wherein the surface of said device is functionalized beforehand with collagen. 19. Method according to claim 14 or 15, comprising the formation of a collagen precipitate in aqueous suspension comprising said hyaluronic acid complex with said glycopeptide antibiotic, its separation and partial drying to obtain a gel. 20.-Antibacterial and osteoinductive implant device according to any one of claims 1 to 10. 12